Nucleosynthesis and astrophysical gamma ray spectroscopy

The HEAO-3 gamma ray spectrometer has provided evidence in the quest for the understanding of complex element formation in the universe with the discovery of Al-26 in the interstellar medium. It has demonstrated that the synthesis of intermediate mass nuclei is currently going on in the galaxy. This discovery was confirmed by the Solar Maximum Mission. The flux is peaked near the galactic center and indicates about 3 solar masses of Al-26 in the interstellar medium, with an implied ratio of Al-26/Al-27 = .00001. Several possible distributions were studied but the data gathered thus far do not allow discrimination between them. It is felt that only the spaceflight of a high resolution gamma ray spectrometer with adequate sensitivity will ultimately resolve the issue of the source of this material

Looking Back: Cyclamate

The second in a series re-evaluating hazards identified in the 1950s and 60s

The Spectrum of Crab Nebula X-Rays to 120 Kev

Counting rate and pulse height distribution spectral data of Crab Nebula telemetered from balloon detector

Functional mapping of the translation-dependent instability element of yeast MATalpha1 mRNA

The determinants of mRNA stability include specific cis-acting destabilizing sequences located within mRNA coding and noncoding regions. We have developed an approach for mapping coding-region instability sequences in unstable yeast mRNAs that exploits the link between mRNA translation and turnover and the dependence of nonsense-mediated mRNA decay on the activity of the UPF1 gene product. This approach, which involves the systematic insertion of in-frame translational termination codons into the coding sequence of a gene of interest in a upf1delta strain, differs significantly from conventional methods for mapping cis-acting elements in that it causes minimal perturbations to overall mRNA structure. Using the previously characterized MATalpha1 mRNA as a model, we have accurately localized its 65-nucleotide instability element (IE) within the protein coding region. Termination of translation 5\u27 to this element stabilized the MATalpha1 mRNA two- to threefold relative to wild-type transcripts. Translation through the element was sufficient to restore an unstable decay phenotype, while internal termination resulted in different extents of mRNA stabilization dependent on the precise location of ribosome stalling. Detailed mutagenesis of the element\u27s rare-codon/AU-rich sequence boundary revealed that the destabilizing activity of the MATalpha1 IE is observed when the terminal codon of the element\u27s rare-codon interval is translated. This region of stability transition corresponds precisely to a MATalpha1 IE sequence previously shown to be complementary to 18S rRNA. Deletion of three nucleotides 3\u27 to this sequence shifted the stability boundary one codon 5\u27 to its wild-type location. Conversely, constructs containing an additional three nucleotides at this same location shifted the transition downstream by an equivalent sequence distance. Our results suggest a model in which the triggering of MATalpha1 mRNA destabilization results from establishment of an interaction between translating ribosomes and a downstream sequence element. Furthermore, our data provide direct molecular evidence for a relationship between mRNA turnover and mRNA translation

NMD monitors translational fidelity 24/7

Nonsense-mediated mRNA decay (NMD) is generally thought to be a eukaryotic mRNA surveillance pathway tasked with the elimination of transcripts harboring an in-frame premature termination codon (PTC). As presently conceived, NMD acting in this manner minimizes the likelihood that potentially toxic polypeptide fragments would accumulate in the cytoplasm. This notion is to be contrasted to the results of systematic RNA-Seq and microarray analyses of NMD substrates in multiple model systems, two different experimental approaches which have shown that many mRNAs identified as NMD substrates fail to contain a PTC. Our recent results provide insight into, as well as a possible solution for, this conundrum. By high-resolution profiling of mRNAs that accumulate in yeast when the principal NMD regulatory genes (UPF1, UPF2, and UPF3) are deleted, we identified approximately 900 NMD substrates, the majority of which are normal-looking mRNAs that lack PTCs. Analyses of ribosomal profiling data revealed that the latter mRNAs tended to manifest elevated rates of out-of-frame translation, a phenomenon that would lead to premature translation termination in alternative reading frames. These results, and related observations of heterogeneity in mRNA isoforms, suggest that NMD should be reconsidered as a probabilistic mRNA quality control pathway that is continually active throughout an mRNA\u27s life cycle

Pbp1p, a factor interacting with Saccharomyces cerevisiae poly(A)-binding protein, regulates polyadenylation

The poly(A) tail of an mRNA is believed to influence the initiation of translation, and the rate at which the poly(A) tail is removed is thought to determine how fast an mRNA is degraded. One key factor associated with this 3\u27-end structure is the poly(A)-binding protein (Pab1p) encoded by the PAB1 gene in Saccharomyces cerevisiae. In an effort to learn more about the functional role of this protein, we used a two-hybrid screen to determine the factor(s) with which it interacts. We identified five genes encoding factors that specifically interact with the carboxy terminus of Pab1p. Of a total of 44 specific clones identified, PBP1 (for Pab1p-binding protein) was isolated 38 times. Of the putative interacting genes examined, PBP1 promoted the highest level of resistance to 3-aminotriazole (\u3e100 mM) in constructs in which HIS3 was used as a reporter. We determined that a fraction of Pbp1p cosediments with polysomes in sucrose gradients and that its distribution is very similar to that of Pab1p. Disruption of PBP1 showed that it is not essential for viability but can suppress the lethality associated with a PAB1 deletion. The suppression of pab1Delta by pbp1Delta appears to be different from that mediated by other pab1 suppressors, since disruption of PBP1 does not alter translation rates, affect accumulation of ribosomal subunits, change mRNA poly(A) tail lengths, or result in a defect in mRNA decay. Rather, Pbp1p appears to function in the nucleus to promote proper polyadenylation. In the absence of Pbp1p, 3\u27 termini of pre-mRNAs are properly cleaved but lack full-length poly(A) tails. These effects suggest that Pbp1p may act to repress the ability of Pab1p to negatively regulate polyadenylation

Dcp2 C-terminal Cis-Binding Elements Control Selective Targeting of the Decapping Enzyme by Forming Distinct Decapping Complexes [preprint]

A single Dcp1-Dcp2 decapping enzyme targets diverse classes of yeast mRNAs for decapping-dependent 5’ to 3’ decay, but the molecular mechanisms controlling selective mRNA targeting by the enzyme remain elusive. Through extensive genetic analyses we uncover cis-regulatory elements in the Dcp2 C-terminal domain that control selective targeting of the decapping enzyme by forming distinct decapping complexes. Two Upf1-binding motifs target the decapping enzyme to NMD substrates, and a single Edc3-binding motif targets both Edc3 and Dhh1 substrates. Pat1-binding leucine-rich motifs target Edc3 and Dhh1 substrates under selective conditions. Although it functions as a unique targeting component of specific complexes, Edc3 is a common component of multiple complexes. Xrn1 also has a specific Dcp2 binding site, allowing it to be directly recruited to decapping complexes. Collectively, our results demonstrate that Upf1, Edc3, and Pat1 function as regulatory subunits of the holo-decapping enzyme, controlling both its targeting specificity and enzymatic activation

Injection mould tool manufacture in less than five days

Using novel rapid prototyping (RP) technology combined with established electroforming tehniques and electro-discharge machining (EDM), injection mould tools have been produced in days rather than weeks. These moulds are manufactured in new silicon-aluminium alloys developed by Osprey Metals, containing 50% or more silicon. The synthesis of these processes shows great potential for use in the rapid tooling sector

Poly(A)-binding proteins: multifunctional scaffolds for the post-transcriptional control of gene expression

Most eukaryotic mRNAs are subject to considerable post-transcriptional modification, including capping, splicing, and polyadenylation. The process of polyadenylation adds a 3' poly(A) tail and provides the mRNA with a binding site for a major class of regulatory factors, the poly(A)-binding proteins (PABPs). These highly conserved polypeptides are found only in eukaryotes; single-celled eukaryotes each have a single PABP, whereas humans have five and Arabidopis has eight. They typically bind poly(A) using one or more RNA-recognition motifs, globular domains common to numerous other eukaryotic RNA-binding proteins. Although they lack catalytic activity, PABPs have several roles in mediating gene expression. Nuclear PABPs are necessary for the synthesis of the poly(A) tail, regulating its ultimate length and stimulating maturation of the mRNA. Association with PABP is also a requirement for some mRNAs to be exported from the nucleus. In the cytoplasm, PABPs facilitate the formation of the 'closed loop' structure of the messenger ribonucleoprotein particle that is crucial for additional PABP activities that promote translation initiation and termination, recycling of ribosomes, and stability of the mRNA. Collectively, these sequential nuclear and cytoplasmic contributions comprise a cycle in which PABPs and the poly(A) tail first create and then eliminate a network of cis- acting interactions that control mRNA function

General decapping activators target different subsets of inefficiently translated mRNAs

The Dcp1-Dcp2 decapping enzyme and the decapping activators Pat1, Dhh1, and Lsm1 regulate mRNA decapping, but their mechanistic integration is unknown. We analyzed the gene expression consequences of deleting PAT1, LSM1, or DHH1, or the DCP2 C-terminal domain, and found that: i) the Dcp2 C-terminal domain is an effector of both negative and positive regulation; ii) rather than being global activators of decapping, Pat1, Lsm1, and Dhh1 directly target specific subsets of yeast mRNAs and loss of the functions of each of these factors has substantial indirect consequences for genome-wide mRNA expression; and iii) transcripts targeted by Pat1, Lsm1, and Dhh1 exhibit only partial overlap, are generally translated inefficiently, and, as expected, are targeted to decapping-dependent decay. Our results define the roles of Pat1, Lsm1, and Dhh1 in decapping of general mRNAs and suggest that these factors may monitor mRNA translation and target unique features of individual mRNAs